At present we do not know to what extent the chain length of folylpolyglutamates serves to direct the flux of one carbon units through competing pathways in mammalian cells. These studies are aimed at providing basic information on the specificities of various folate-dependent enzymes for the number of glutamyl residues in the polyglutamyl side chain of folate cosubstrates and inhibitors. Such comparisons require that all the enzymes studied be isolated from a single source, and we have chosen pig liver. In this way, the differences in specificity which may be observed will not be attributable to interspecies differences. We have already performed these studies on methylenetetrahydrofolate reductase and serine hydroxymethyltransferase from pig liver. We would now llike to extend these studies to thymidylate synthase and methylenetetrahydrofolate dehydrogenase from pig liver. We plan to mesure the decrease in free energy associated with the binding of folylpolyglutamate cosubstrates or inhibitors to these two enzymes, and to characterize the way in which that free energy of binding is expressed in catalysis. These studies should increase our understanding of the factors which regulate one carbon metabolism in mammalian cells and they should provide information which will be useful for the evaluation of inhibitors of these folate-dependent enzymes which are substrate analogues, such as fluorodeoxyuridylate (FdUMP).